Mammalian hemoglobins are composed of two pairs of chemically distinct subunits, designated alpha and beta in the case of adult human hemoglobin. Since the primary structures of the two types of subunits are different, the alpha and beta chains are neither expected nor observed to make an equal contribution to the ligand binding properties of hemoglobin. The structural causes of these differences in reactivities of the chains will be investigated by varying the size of the heme ligand, by substituting unnatural hemes for protoheme, and by examining the properties of minor hemoglobin components and hemoglobins from different species. Studies of the differential release of anions and protons associated with ligand binding to the individual chains are also proposed. Finally, the reaction of the isolated alpha and beta chains to form native tetrameric hemoglobin will be examined. The majority of the proposed exeriments involve kinetic measurements using rapid mixing stopped-flow techniques. These studies are designed to provide new information concerning the relative contributions of the alpha and beta chains to the overall physical-chemical properties of hemoglobin. Information of this type will allow the correlation of functional data with more specific structural intermediates (i.e. the alpha and beta chains), may provide explanations for certain apparent anomalies in experimental data, and is necessary for an understanding of the evolutionary development of hemoglobin, of the importance and function of minor hemoglobin components, and of the clinical expression and consequences of certain hereditary hemoglobinopathies. BIBLIOGRAPHIC REFERENCES: Olson, J.S., Spectral Transitions of Methemoglobin, Federation Proceedings 34, 603 (1975). Wiedermann, B.L., and Olson, J.S., Acceleration of Tetramer Formation by the Binding of Inositol Hexaphosphate to Hemoglobin Dimers, J. Biol. Chem. 250, 5273-5275, (1975).